Protein kinases play a critical role in cell signalling pathways by catalyzing transfer of the γ-phosphoryl group from ATP to the hydroxyl groups of protein side chains1. Approximately 2% of eukaryotic genes encode protein kinases making this one of the largest protein superfamilies. Because of their importance in contributing to a variety of pathophysiologic states including cancer, inflammatory conditions, autoimmune disorders, and cardiac diseases, there have been intense efforts to develop specific protein kinase inhibitors as biological tools and as therapeutic agents2. Considerable success has been achieved developing potent and selective nucleotide-based analog inhibitors that interact with individual protein kinases at their nucleotide binding sites, and several compounds are in early phases of human clinical trials2. In general, the protein substrate binding site has not been exploited for inhibitor design3. Moreover, mechanism-based approaches to generating protein kinase inhibitors have been unsuccessful4-8. This situation stands in contrast to many other important enzyme classes, such as the proteases, where consideration of enzyme mechanism and structure has led to potent inhibitors, some of which are clinically useful drugs9.
Protein kinases follow ternary complex kinetic mechanisms in which direct transfer of the phosphoryl group from ATP to protein substrate occurs10. For such mechanisms, designing covalently linked bisubstrate analogs can be a powerful approach toward potent enzyme inhibitors9. However, previous attempts to employ this strategy with protein kinases have met with mixed results4, 8, 11. A sophisticated effort reported by Gibson and colleagues linked ATP directly to the serine oxygen of a protein kinase A (PKA) peptide substrate (kemptide) to generate an inhibitor (1, FIG. 1a)8. Compound 1 was a weak inhibitor with an IC50 of 226 mM (compared to Km (ATP) of 10 mM and Km (kemptide) of 15 mM) which was competitive versus ATP but non-competitive versus peptide substrate. While not providing all the desired features of a bisubstrate analog, these results suggest that improvements in geometry and electronic character around the atoms equivalent to the entering nucleophile and reacting phosphate would benefit inhibitor design. There is a need in the art for kinase inhibitors which are stronger than those currently available.